Waste water treatment

ABSTRACT

The present invention relates to wastewater treatment in general and to methods of controlling odors and degrading compounds contained in wastewater in particular.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority or the benefit under 35 U.S.C. 119 ofU.S. provisional application no. 60/911,308 filed Apr. 12, 2007, thecontents of which are fully incorporated herein by reference.

CROSS-REFERENCE TO DEPOSITED MATERIALS

The present application refers to deposited microorganisms. The contentsof the deposited microorganisms are fully incorporated herein byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to wastewater treatment in general and tomethods of controlling odors, reducing chemical oxygen demand (COD), anddegrading compounds contained in wastewater in particular.

2. Description of Related Art

The main chemical compounds in wastewater are nitrogen, phosphorus,fats, oils and grease.

Objectionable odors are caused by a variety of substances typicallypresent in wastewater. These include sulfur and several sulfurcontaining compounds including hydro sulfuric acid, sulfuric acid,mercaptans (R—SH) including especially methyl and dimethyl mercaptans,and dimethyl disulfide (DMDS); numerous organic acids includingpropionic acid, acetic acid, butyric acid, isovaleric acid; ammonia;urea; and various terpenes including carene, pinene, limonene. Thesesubstances most frequently lead to noticeable odors under anaerobicconditions.

Octel Gamlen has sold a wastewater treatment composition comprising astrain of each of Mucor hiemalis, Trichoderma atroviride, Paecilomycesvariottii, and Aspergillus niger.

U.S. Pat. No. 7,160,458 discloses a method for purifying process waterfrom a kerosene desulfurization plant comprising adding bacterialspecies.

It is an object of the invention to provide an improved wastewatertreatment composition.

SUMMARY OF THE INVENTION

The present invention is directed to wastewater treatment compositionscomprising a strain of Mucor racemosus, Paecilomyces lilacinus,Aspergillus ustus or Trichoderma inhamatum (anamorph is Hypocreagelatinosa).

In another embodiment, the present invention relates to methods for thetreatment of wastewater comprising adding to the wastewater a strain ofMucor racemosus, Paecilomyces lilacinus, Aspergillus ustus orTrichoderma inhamatum (anamorph is Hypocrea gelatinosa).

The present invention also relates to a process of degrading compoundscontained in a wastewater and biologically pure cultures of one or moremicrobial strains.

DETAILED DESCRIPTION OF THE INVENTION Wastewater Treatment Compositions

The present invention is directed to wastewater treatment compositionscomprising a strain of Mucor racemosus, Paecilomyces lilacinus,Aspergillus ustus or Trichoderma inhamatum and to methods for thetreatment of wastewater comprising adding to the wastewater a strain ofMucor racemosus, Paecilomyces lilacinus, Aspergillus ustus orTrichoderma inhamatum.

Strains of Mucor racemosus, Paecilomyces lilacinus, Aspergillus ustusand Trichoderma inhamatum strains were deposited for patent purposesunder the terms of the Budapest Treaty at the NRRL USDA-ARS PatentCulture Collection, 1815 N. University Street, Peoria, Ill. 61604. Thedeposits were made on Mar. 20, 2007 by Novozymes Biologicals Inc. andwere accorded deposit numbers:

-   Mucor recemosus NRRL 50031-   Paecilomyces lilacinus NRRL 50032-   Aspergillus ustus NRRL 50033-   Trichoderma inhamatum NRRL 50034

In a preferred embodiment, the wastewater composition comprises a strainof Mucor racemosus.

In another preferred embodiment, the wastewater composition comprises astrain of Paecilomyces lilacinus.

In another preferred embodiment, the wastewater composition comprises astrain of Aspergillus ustus.

In another preferred embodiment, the wastewater composition comprises astrain of Trichoderma inhamatum.

In another preferred embodiment, the wastewater composition comprises astrain of Mucor racemosus and Paecilomyces lilacinus.

In another preferred embodiment, the wastewater composition comprises astrain of Mucor racemosus and Aspergillus ustus.

In another preferred embodiment, the wastewater composition comprises astrain of Mucor racemosus and Trichoderma inhamatum.

In another preferred embodiment, the wastewater composition comprises astrain of Paecilomyces lilacinus and Aspergillus ustus.

In another preferred embodiment, the wastewater composition comprises astrain of Paecilomyces lilacinus and Trichoderma inhamatum.

In another preferred embodiment, the wastewater composition comprises astrain of Aspergillus ustus and Trichoderma inhamatum.

In another preferred embodiment, the wastewater composition comprises astrain of Mucor racemosus, Paecilomyces lilacinus, and Aspergillusustus.

In another preferred embodiment, the wastewater composition comprises astrain of Mucor racemosus, Paecilomyces lilacinus, and Trichodermainhamatum.

In another preferred embodiment, the wastewater composition comprises astrain of Paecilomyces lilacinus, Aspergillus ustus and Trichodermainhamatum.

In another preferred embodiment, the wastewater composition comprises astrain of Mucor racemosus, Paecilomyces lilacinus, Aspergillus ustus andTrichoderma inhamatum.

The strains may be wild-type or mutant strains.

In a preferred embodiment, the composition comprises the microorganismat a concentration of 1×10² to 1×10⁹ colony forming units (CFU)/mL,preferably 1×10⁴ to 1×10⁹ colony forming units (CFU)/mL. When thecomposition contains more than one microorganism, each microorganism ispresent at a concentration of 1×10⁴ to 0.5×10⁹ colony forming units(CFU)/mL.

In another preferred embodiment, the composition further comprisesnutrients for the microorganism(s). For example, the nutrients may be aninorganic phosphorus compound, particularly a soluble phosphate or anortho phosphate, preferably, phosphoric acid, mono, di, or tri sodiumphosphate, or diammonium phosphate. In addition, the nutrients may beammonia (NH₃) or an ammonium (NH₄ ⁺) salt, preferably anhydrous ammonia,ammonia-water solutions, ammonium nitrate, or diammonium phosphate. Thenutrients may also be trace metals, preferably aluminum, antimony,barium, boron, calcium, cobalt, copper, iron, lead, magnesium,manganese, molybdenum, nickel, strontium, titanium, tin, zinc, and/orzirconium.

In another preferred embodiment, the composition further comprises asugar selected from the group consisting of arabinan, arabinose,cellulose, fructose, galactan, galactose, glucan, glucose, mannan,mannose, sucrose, xylan, and xylose, or wood fiber, wood pulp, or otherpulping byproducts. Preferably, the composition comprises the sugar at aconcentration between 100 and 400 mg/L, when the sugar is amonosaccharide and a concentration between 8,000 and 15,000 mg/L, whenthe sugar is a polysaccharide.

The wastewater to be subjected to the process of this invention maycontain sufficient nutrients, e.g., nitrogen and phosphorus, forculturing without the need for any additional source of nitrogen orphosphorus being added. However, in the event the wastewater isdeficient in these components, nutrients can be added to the wastewater.For example, phosphorous can be supplemented, if necessary, by additionof a phosphorous source such an inorganic phosphorus compound,particularly a soluble phosphate or an orthophosphate, preferably,phosphoric acid, mono, di, or tri sodium phosphate, or diammoniumphosphate, to achieve a phosphorus level in the wastewater of about 1ppm or more per 100 BOD₅. Similarly, a nitrogen source, such as ammonia(NH₃), urea, or an ammonium salt, preferably anhydrous ammonia,ammonia-water solutions, ammonium nitrate, or diammonium phosphate, canbe added to achieve an available nitrogen content of at least about 10ppm or more per 100 BOD₅.

In another embodiment, the nutrients comprise trace metals, preferablyaluminum, antimony, barium, boron, calcium, cobalt, copper, iron, lead,magnesium, manganese, molybdenum, tin, or zinc.

Methods for Treating Wastewater

The present invention also relates to methods for treating wastewaterwith a wastewater treatment composition of the present invention.

The wastewater treatment process of the present invention may reduceodor, as well as degrade compounds contained in wastewater such asbutanoic acid, 2-methylphenol, heptanoic acid, nonanoic acid,5-bromothiophene-2-carboxamide, isoxazolidine and2-methyl-1-nitropropane.

Other odor-causing compounds which may be degraded by a wastewatertreatment composition of the present invention are hydrogen sulfide,trimethylamine, methanethiol, butanoic acid, 3-methylbutanoic acid,pentanoic acid, 4-methylphenol, dimethylsulfide, dimethyldisulfide,propanoic acid, acetic acid, 2-methylpropanoic acid, indole, and3-methyl-1H-indole.

The wastewater treatment compounds may also reduce chemical oxygendemand (COD) of wastewater.

The strains used in the present invention can be cultured in wastewaterfrom, e.g., a pulp or paper mill either using a batch process, asemi-continuous process or a continuous process, and such is culturedfor a time sufficient to degrade compounds present in the wastewater andremove them or break them down into components capable of being degradedby other organisms normally found in biological wastewater treatmentsystems.

The microbial strains of this invention can be employed in ion exchangeresin treatment systems, in trickling filter systems, in carbonadsorption systems, in activated sludge treatment systems, in outdoorlagoons or pools, etc.

Basically, all that is necessary is for the microorganism(s) to beplaced in a situation of contact with the wastewater effluent from apulp or paper mill. In order to degrade the material present in thewastewater, the wastewater is treated with the organism(s) at atemperature between 15° C. and 45° C., preferably between 20° C. and 45°C., more preferably between 18° C. and 37° C., and most preferablybetween 30° C. and 35° C. Desirably, the pH is maintained in a range of4 and 10, preferably 4.5 to 8.5. The pH can be controlled by monitoringof system and an addition of appropriate pH adjusting materials toachieve this pH range.

In general, the treatment is conducted for a sufficient time to achievethe reduction in odor or degradation of compounds desired and, ingeneral, about 24 hours to about 8 weeks or longer, although this willdepend upon the temperature of culturing, the liquor concentration andvolume to be treated and other factors. In a preferred embodiment, thewastewater is treated with the microorganism(s) for between 2 hours and14 days, preferably between 2 hours and 5 days.

The treatment can be conducted under aerobic or anaerobic conditions.When aerobic conditions are used, the treatment is conducted at adissolved oxygen concentration of between 0.5 and 7.0 milligrams perliter. These conditions can be simply achieved in any mannerconventional in the art and appropriate to the treatment system designbeing employed. For example, air can be bubbled into the system, thesystem can be agitated, a trickling system can be employed, etc. In anaerobic process, the treatment is done at a REDOX potential between −200mV and 200 mV, preferably between 0 mV and 200 mV. When anaerobicconditions are used, the treatment is done at a REDOX potential between−550 mV and −200 mV.

Normally aerobic measures are undertaken to reduce colorants andbiochemical oxygen demand (BOD) in wastewater. Aerobic technologiesinclude trickling filter, activated sludge, rotating biologicalcontactors, oxidation ditch, sequencing batch reactor and evencontrolled wetlands.

An anaerobic or anaerobic-friendly type of technology can also be usedfor treating the wastewater. Anaerobic technologies currently availableare high-rate systems including continuous-flow stirred tank reactors,contact reactors, upflow sludge blankets, anaerobic filters (upflow anddownflow), expanded or fluidized bed and two-stage systems that separatethe acid-forming and the methane-forming phases of the anaerobicprocess.

Aerobic and anaerobic processes can be combined into a treatment system.Anaerobic treatment may be used for removing organic matter in highconcentration streams, and aerobic treatment may be used on lowerconcentration streams or as a polishing step to further remove residualorganic matter and nutrients from wastewater.

In a preferred embodiment, the wastewater treatment comprises 1-5cycles, preferably 1 cycle or two cycles, of treatment with themicroorganism(s). Preferably, each cycle comprises alternating aerobicand anaerobic treatments. More preferably, the first cycle is conductedunder aerobic conditions. In a preferred embodiment, the cycles areconducted in a sequencing batch reactor. In another preferredembodiment, the process further comprises adding an alkali betweencycles.

Preferably, the wastewater is a pulp and paper mill wastewater such asstrong or concentrated pulp mill wastewater, weak black liquor, acidstage bleach plant filtrate, or alkaline stage bleach plant filtrate.Other types of wastewaters that might be treated include cleaning andlaundry wastewaters, food processing wastewaters, and industrial processwaters such as vegetable oil extractions or waste materials havingfiber-containing by-products.

The process also can be used to treat waste from chemical colorseparation processes commonly used in wastewater treatment, includinggravity clarifiers, gas flotation units, or in filtration processes suchas membrane processes.

In another preferred embodiment, the ratio of solids to liquid waste isbetween 1:50 to 10:1 preferably 1:10 to 5:1.

In another preferred embodiment, the wastewater passes through woodfibers at anaerobic conditions, particularly in a packed biologicalreactor or column, an artificial wetland, or an anaerobic sequencingbatch reactor (AnSR). Alternatively, the wastewater passes through amass comprising waste wood fiber from a pulp & paper process, lime, andfly ash. Preferably, the wastewater passes through wood fiber togetherwith cellulosic fiber, plastic, powdered or ceramic media. The rate ofthe wastewater is preferably 0.05-1 liter wastewater/day per kilogram ofwet wood fiber mass.

In a most preferred embodiment, wood fiber is used as a biologicalmedium at anaerobic conditions, comprising one or more of the followingsteps of: (a) sequencing batch reactors, (b) a facultative lagoon or astabilization basin, (c) an activated sludge system, (d) coagulation andflocculation followed by settling, and (e) filtration.

The wastewater may be treated with the microorganism(s) in the presenceof an electron acceptor, particularly chloroethanes, chloroform,chlorolignins, chloromethanes, chlorophenols, humates, lignin, quinines,or sulfonated lignins.

The microorganisms of the present invention can be employed alone or incombination with conventionally means for treating wastewater, e.g.,chemical (e.g., alum, ferric, lime or polyelectrolytes), biological(e.g., white rot fungus), and physical processes (e.g., ultrafiltration,ion exchange and carbon absorption).

In the above manner, organic compounds which are present in suchwastewater streams, can be advantageously treated to provide treatedwastewater suitable for discharge after any additional conventionalprocessing such as settling, chlorination, etc. into rivers and streams.

Formulations

The individual fungal strains or the blends noted above can be providedon the original media material used to culture the strains, or they canbe removed from the original growth substrate by various physicalmechanisms and reblended on a separate substrate or addition to achievethe desired concentration for a given application. For example, fungalspores or other discreet propagules might be removed by sonication,washing, or substrate breakdown, followed by a concentration strep suchas sieving, centrifugation, or other size-exclusion techniques familiarto those skilled in the art. Such separated and/or concentrated,propagules may be either blended and applied directly, or placed on aseparate substrate for application. In this way, undesirable physicalproperties of the original growth substrate, such as lack of solubility,or poor liquid pumping characteristics, can be improved and the productmay be more readily, easily, or economically applied. In a preferredembodiment, fungal spores of the Mucor racemosus strain can be removedfrom the growth media by sonication, concentrated by sieving andcentrifugation, then combined with one or more of the other strains, toprovide a liquid concentrate suspension that may be automaticallydelivered by pumping to the desired wastewater reaction area. Varioussuspension agents and/or surfactants could be added to aid pumping orreduce settling of the concentrated fungal propagule blend.

Cultures

The present invention also relates to a biologically pure culture of astrain of Mucor racemosus, Paecilomyces lilacinus, Aspergillus ustus orTrichoderma inhamatum.

The following examples are given as exemplary of the invention butwithout intending to limit the same. Unless otherwise indicated herein,all parts, percents, ratios and the like are by weight.

EXAMPLES Example 1 Materials and Methods: Media and Substrates:

Pulp and Paper Mill waste streams: The wastewater used in the laboratorystudies were obtained from various pulp and paper mills in the U.S. andFrance. The waste stream material was brought to pH 7.8 by the additionof a nutrient (N&P) amended media based on SSC (see the table below).

SAMPLE A SAMPLE B packaged in 0.5 kg water-soluble sachet packaged boxesof bulk powder 2 fungal strains: 3 fungal strains: Mucor hiemalis Mucorhiemalis Trichoderma atroviride Aspergillus niger Paecilomyces variottiiCarrier of fungi: maltodextrine Carrier of fungi: maltodextrineAdditional medium (excipient): Additional medium (excipient):lithothamne wheat bran Dosage rate: 2 g of A per kg Dosage rate: 2 g ofB per kg of COD of COD Total count: 1 × 10⁴ propagules Total count: 1 ×10⁴ propagules per gram per gram

Industrial Waste Stream: The waste stream was used as received in thisstudy except where noted. The waste stream comes from a site thatproduces architectural and functional coatings and plastics additives(impact modifiers and processing aids) and has regular problems withlatex. The pH of the waste stream was found to be 8.9 and it was alsofound to contain a number of protozoa. The soluble COD was 477±8 mg/L.The waste stream was reported to have an average influent COD of 1400mg/L with an effluent COD of 400 mg/L. The waste stream as received hada low COD which was not due to COD loss during transportation, butinstead was due to the low COD of the waste stream when collected.Microscopic examination of the waste revealed protozoa present in thesample.

Laundry Waste Stream: The laundry waste stream was used as received andwas from a denim fabric factory with 0.2% Aquazyme Ultras 1200 L. Theinitial pH of this waste stream was 5.6.

Waste stream preparation: Sterilization of the waste stream where notedwas accomplished by filtration. The waste stream was first centrifugedat 12,000× G for 60 minutes. This material was then passed in 100-150 mLaliquots over Whatman 934-AH filters, followed by filtration throughWhatman GF/F filters, followed by filtration though a Gelman Sciences0.45 micro-m Metricel membrane filter. Final sterilization wasaccomplished by filtering though a pre-sterilized Nalgene filtrationunit equipped with a 0.22 micro-m membrane filter.

Nutrent Additions: The components of the nutrient media are listed inthe table below. These were prepared as a ten fold concentrate and addedto the waste stream to give the final concentrations listed in thefollowing table.

Nutrient Media Component (g/L) K₂HPO₄ 2.0 KH₂PO₄ 3.06 NH₄Cl 0.8MgSO₄•7H₂O 0.2 CaCl₂•H₂O 0.01 ZnSO₄ 0.000140 MnSO₄•H₂O 0.000084NaMoO₄•2H₂O 0.000024 FeSO₄•7H₂O 0.000028 CuSO₄•5H₂O 0.000025 CoCl₂•6H₂O0.000024 Adjust pH to 7.5 with KOH

Culture Conditions: Incubations of the pulp mill waste were carried outin sterile 150 mL serum vials containing 10 mL of filter-sterilizedwaste. The tops of the vials were covered with “steam paper” to allowfor oxygen transfer. Incubations were carried out at 30° C. Thereactions with the Laundry waste and the Industrial waste were carriedout in 250 mL shake flask with 50 or 100 mL of filter-sterilized waste.

Inoculum Preparation: For experiments involving the formulated Sample A,Sample B, or NZB—C sample product, to 0.1-1 grams of product was addedsterile phosphate buffer to give a final concentration of 0.11 gramsproduct/ml of buffer. This was then agitated on a wrist action shakerfor 30 minutes. Except were noted, the products were added to a finalconcentration of 1 gram/300 mL waste stream.

Dry product production: The dry products used in this study had thecharacteristics listed in table below. The sample products were made byfirst growing the isolated fungus in 110 gram lots on a mediumconsisting of 50 grams of rice hulls, 50 grams bran and 10 grams starch.To this material was added 100 mL of 50% potato dextrose agar (PDA) formoisture. The material was autoclaved and inoculated with fungal myceliaand spores from pre-grown PDA plates. With the exception of P.chrysosponum, all incubations were initially carried out at 25° C. for7-10 days in a humidified growth chamber in 190×100 mm glass dishes. P.chrysospoium was initially cultured at 39° C. At the end of the initialincubation, the cultures were removed from the humidified growth chamberand allow to air dry at room temperature for an additional 5-7 days.Final drying was accomplished under reduced pressure in a lyophilizer.

The raw material was then subjected to hydration and serial dilution todetermine the number of propagules/gram using standard laboratoryprocedures. With the exception of P. chrysosporium, all fungi had yieldsof 1.0×10⁹ to 1.0×10⁷ propagules/gram. P. chrysoporium yielded 4.0×10³propagules/gram.

Concentration (propagules/gram) of Organisms in NZB-C Dry Formulatedsample SINGLE ORGANISMS NZB-C Mucor racemosus 2.0 × 10⁵ (range 0.1 to 10× 10⁵) Aspergillus ustus 2.9 × 10⁵ (range 0.1 to 10 × 10⁵) Paecilomyceslilacinus 1.3 × 10⁵ (range 0.1 to 10 × 10⁵) Trichoderma inhamatum 3.0 ×10⁵ (range 0.1 to 10 × 10⁵) Total 9.2 × 10⁵ (range 0.4 to 40 × 10⁵)

COD Assay. At the indicated time, soluble COD was determined by Method5220C (Standard Methods). All material was centrifuged at 13,000× G for20 minutes to remove particulates. All data represents soluble COD andunless noted the mean of three determinations ± one standard deviationunit.

Results:

Experiments with Pure Cultures: In order to assess the role ofindividual fungi, pure and mixed cultures of the fungi were incubatedwith waste from a pulp and paper mill “strong pond.” The species andconcentration of each fungus are listed in the table below. It isimportant to note that for the fungal consortia, the competitor fungiwere added in greater concentrations.

ORGANISMS AND THEIR CONCENTRATIONS USED FOR “PURE” CULTURE STUDIESConsortia Consortia Single Organisms Propagule/mL Name CompositionPropagule/mL Mucor racemosus 1.8 × 10⁴ Sample A Hypocrea gelatinosa 2.8× 10⁵ Trichoderma 1.4 × 10⁵ atroviride Phenerochaete 1.0 × 10⁴ Mucorhiemalis 1.8 × 10⁷ chrysosporium Aspergillus ustus 3.2 × 10⁴ Sample BPaecilomyces lilacinus 1.4 × 10⁵ Mucor hiemalis 8.0 × 10⁵ Aspergillussp. 5.4 × 10⁷ Paecilomyces 2.7 × 10⁴ variottii Aspergillus niger 3.1 ×10⁴

The degradation of the waste from the primary clarifier from a pulp andpaper mill is shown in Table 1. The NZB—C sample demonstrated betterremoval of COD than the competitor samples.

TABLE 1 Laboratory pulp and paper mill wastewater assessment; 9 Dayspost-treatment Total COD % COD Reduction Treatment (mg/L) vs. ControlControl 310 0 Sample A 285 5 Sample B 270 13 NZB-C 225 28

Example 2

For this experiment, a total of 60 150 ml serum vials were used and werecapped with butylated rubber stoppers. Each sample was done intriplicate. Vials were then sterilized by autoclave @ 121° C. for 30min.

The wastewater was obtained from France. Due to the large amount ofparticulate matter suspended in the samples, the middle and outletwastewaters were filtered. Filtering was accomplished by usingsuccessively smaller filter sizes until the final filter size was 0.2micro-m. A Whatman 934-AH filter (1.5 micro-m) was used first to removelarge particulate matter in the samples. Then a Fisherbrand 0.45 micro-mMembrane MCE filter (catalog #09-719-2E) and Fisherbrand 0.2 micro-mMembrane MCE filter (catalog #09-719-2B) was used successively toachieve the desired filtration size.

After filter-sterilizing the middle and outlet waste, a 10 ml volume ofwastewater was added to each of the vials under a laminar flow hood.Each vial was supplemented with 1×SSC to aid in fungal growth.

A 1×SSC nutrient media was prepared as follows:

1xSSC Nutrient Media Component g/L K₂HPO₄ 2.0 KH₂PO₄ 3.06 NH₄Cl 0.8MgSO₄•7H₂O 0.2 CaCl₂•H₂O 0.01 ZnSO₄ 0.000140 MnSO₄•H₂O 0.000084NaMoO₄•2H₂O 0.000024 FeSO₄•7H₂O 0.000028 CuSO₄•5H₂O 0.000025 CoCl₂•6H₂O0.000024 Adjust pH to 7.5 with KOH

1×SSC was added to each of the serum vials. To prepare the inocula fromthe two dry products (NZB—C, a consortium of strains of Mucor racemosus,Paecilomyces lilacinus, Aspergillus ustus, and Trichoderma inhamatumdeposited with NRRL and accorded deposit nos. NRRL 50031, NRRL 50032,NRRL 50033, and NRRL 50034, respectively, and Bi-Chem1005PP, a bacterialproduct from Novozymes Biologicals) 2.5 g of the product was added to 25ml of sterile phosphate buffer in a sterile test tube which was thenagitated on a wrist action shaker for 15 min. To prepare the inoculafrom a single fungal strain culture, a plate of each strain was obtainedand the mixture of spores and mycelia were scraped from the surface ofthe plate with a sterile cotton swab. The swab was then submerged in 99ml 0.3 mM phosphate buffer with 2 mM MgCl₂, pH 7.4 and vigorouslyagitated for 15 min to release the spores/mycelia into the buffer. Dueto the absorbent properties of the cotton swabs, some volume of thephosphate buffer was lost. The volume was brought back to 10 ml aftercompletion of the swabbing of the plate. In the case of Mucor, twoplates of Mucor grown on PDA were cut into 8 sections and were added to99 ml of phosphate buffer. After agitation, the inocula (dry blend orscrapped from individual culture) was set to stand for 10 min allowingthe particulate matter to settle, then 100 microliters of eachsuspension was taken and used to inoculate the corresponding serum vialscontaining the wastewater and 1×SSC. Vials were then incubated at 35° C.for 14 days.

GC/MS With SPME Analysis of Treated Wastewater from Norampac

In order to detect specific compounds present in the wastewater, GC/MS(Gas chromatography/Mass spectrometry) analysis of the samples wasconducted using SPME (Solid-Phase Microextraction). After COD analysisof each sample, the remainder of the sample (roughly 9 ml) was added toa 20 ml head space vial. A Divinylbenzene/Carboxen/Polydimethylsiloxane(DVB/CAR/PDMS) fiber (grey holder) was selected. The samples were heatedto 60° C. for 5 minutes while being agitated at 320 rpm for 5 secondsand off for 30 seconds to aid in volatilization. Samples were thenextracted for 30 minutes at 60° C. while being agitated as prescribedbefore. Samples were desorbed for 1 minute at 250° C. The split ratiowas set to 1:2 and the septum purge was set at 2.5 ml/min. The sampleswere analyzed on a SPB-1 sulfur column because it was believed thatsulfurous compounds contributing to the odor would be present in thesamples. After desorbtion, the column was held at 40° C. for 3 minutes.Then the temperature was raised to 125° C. at a rate of 4° C./min. Thiswas followed by a more rapid ramp of 25° C./min to 200° C. The massspectrometer was set to scan from 45-1000 amu after a fresh tune.

GC/MS With SPME Analysis of Treated Wastewater from Norampac Trial 2

After it was confirmed that compounds can be detected using GC/MS withSPME, a second trial was set up in order to show degradative ability ofthe unknown compounds in the wastewater. It was hypothesized that ifNZB—C was capable of degradation then it would show up in thechromatograms generated from untreated vs. treated samples. For example,a peak with a retention time of 16.382 minutes in an untreated sampleshould theoretically show up at 16.382 minutes in the treated sample.One could analyze the areas of these peaks and draw a conclusion as tothe degradative ability of the fungal product. For this particularstudy, outlet water from Norampac was filtered through a 0.2 micro-msterile filter, and then added to each respective 20 ml headspace vile.This was done in triplicate. SSC was added to each vial to bring the SSCconcentration to 1×. The following recipe was used for this:

SSC Trace Minerals Solution for 10xSSC preparation Component mg/LZnSO₄•7H₂O 140.0 MnSO₄•H₂O 84.0 NaMoO₄•2H₂O 24.0 FeSO₄•7H₂O 28.0CuSO₄•5H₂O 25.0 CoCl₂•6H₂O 24.0 DI Water To 1000 mL

The media components for ten fold concentrate of SSC are shown in thefollowing table. A white precipitate will form and is normal. The 10×medium is shaken then diluted 10 fold before use into distilled water.The final medium may be autoclaved, but filter sterilization ispreferred.

10X SSC Nutrient Medium Component g/L K₂HPO₄ 20.0 KH₂PO₄ 30.6 NH₄Cl 8.0MgSO₄•7H₂O 2.0 CaCl₂•H₂O 0.1 FeCl₃ 0.05 SSC₃ Trace Minerals Solutionfound in Table 2 10.0 mL DI Water To 1000 mL Adjust pH to 7.0-7.5 withKOH

To prepare the treated samples, a 10% solution of NZB—C was preparedusing 2.5 g of NZB—C dry product added to 25 ml of sterile phosphatebuffer housed in a 50 ml test tube. The tube was the agitated for 15minutes. Once agitation was complete and the bran was allowed to settleto the bottom, 100 microliters were taken from the liquid layer abovethe bran. This was used to inoculate each of the respective treatedvials. Vials were then capped and incubated at 30° C. for 14 days andthen read using the GC/MS protocol prescribed in the previous sectionentitled, “GC/MS with SPME Analysis of Treated Wastewater fromNorampac.”

GC/MS With SPME Analysis of Treated Wastewater from Norampac Trial 2

After confirming that certain compounds could be detected using GC/MSheadspace analysis with SPME, a comparison of the degradation of thecompounds by simple peak comparison between treated and untreatedsamples was made. Three peaks were isolated for this comparison study.The identity of these peaks was provided by the internal compoundlibrary of the Shimadzu GC/MS system used (GCMS-QC2010S). The resultsare provided in Table 2. It is evident that NZB—C is able to degradethese detected compounds.

TABLE 2 Assessment of NZB-C Activity against Waste Compounds in Pulp andPaper Mill Middle and Outlet Wastewater using GC/MS with SPME. AmountAmount % NZB-C Remaining - Remaining - Degradation Control (Peak NZB-CImprovement area units) (Peak area units) vs. Control Compound in MiddleWaste Water 5-bromo-thiophene-2- 3,058 1,721 43.7 carboxamideIsoxazolidine 9,945 8,860 10.9 Dodecamethyl- 13,064 11,987 8.2cyclohexasiloxane Compound in Outlet Waste Water 5-bromo-thiophene-2-6,800 1,822 73.2 carboxamide Isoxazolidine 10,779 7,177 33.4 2-methyl-1-4,507 1,228 72.7 nitropropane

The results show that NZB—C degrades 5-bromothiophene-2-carboxamide,isoxazolidine and 2-methyl-1-nitropropane.

Example 3

A field assessment of the ability of the NZB—C fungal consortium toreduce odor-causing and certain recalcitrant waste compounds in a Pulpand Paper mill lagoon treatment facility in Bonduelle, France wasundertaken. NZB—C material, prepared as described above, was added at arate of 1.5 g NZB—C per 1.0 Kg of total COD present into a treatmentlagoon with a flow rate of 7,500 m³/day. A similar but separate lagoonon location was not treated with NZB—C and served as a control. Sampleswere taken at the lagoon outlet at the initiation of the experiment (Day0) and at Day 23. These were assessed using the GC/MS with SPMEanalytical method described in Example 1. The results are provided inTable 3, and indicate that considerable and significant reduction incertain odor-associated compounds occurred in this time period.

TABLE 3 Field Assessment of NZB-C Activity against Odor-AssociatedCompounds in Pulp and Paper Mill Outlet Wastewater using GC/MS withSPME. Amount Amount % NZB-C Odor Associated Degraded - Degraded -Degradation Compounds Control (Peak NZB-C (Peak Improvement (Outletwater) area units) area units) vs. Control Butanoic Acid 837,019 894,7486.90 2-Methylphenol 192,031 401,809 109.24 Heptanoic Acid 6,275,5738,593,919 36.94 Nonanoic Acid 708,757 1,410,612 99.03

1-48. (canceled)
 49. A process for treating wastewater, comprisingadding to the wastewater a wastewater treatment composition comprising astrain of Mucor racemosus, Paecilomyces lilacinus, Aspergillus ustus orTrichoderma inhamatum.
 50. The process of claim 49, wherein thewastewater treatment composition further comprises a carbohydrateselected from the group consisting of arabinan, arabinose, cellulose,fructose, galactan, galactose, glucan, glucose, mannan, mannose,sucrose, xylan, and xylose, or wood fiber, wood pulp, or other pulpingbyproducts.
 51. The process of claim 49, wherein the wastewatertreatment composition comprises the carbohydrate at a concentrationbetween 100 and 400 mg/L, when the carbohydrate is a monosaccharide anda concentration between 8,000 and 15,000 mg/L, when the carbohydrate isa polysaccharide.
 52. The process of claim 49, wherein the treatment isperformed under aerobic conditions.
 53. The process of claim 49, whereinthe treatment is a batch, semi-continuous or continuous treatment. 54.The process of claim 53, wherein the treatment is done in cycles in asequencing batch reactor.
 55. The process of claim 54, furthercomprising addition of an alkali between cycles.
 56. The process ofclaim 49 wherein the treatment of the wastewater comprises 1-5 cycles oftreatments with the wastewater treatment composition.
 57. The process ofclaim 56, wherein each cycle comprises alternating aerobic and anaerobictreatments.
 58. The process of claim 57, wherein the first treatment isanaerobic.
 59. The process of claim 49, further comprising addingnutrients to the wastewater.
 60. The process of claim 49, wherein thewastewater is a pulp and paper mill wastewater.
 61. The process of claim49, comprising passing the wastewater through wood fibers at anaerobicconditions in a packed biological reactor or column, an artificialwetland, or an anaerobic sequencing batch reactor (AnSR).
 62. Theprocess of claim 49 comprising passing the wastewater through a masscomprising waste wood fiber from a pulp & paper process, lime, and flyash.
 63. The process of claim 62, wherein the wastewater is passedthrough wood fiber at a rate of 0.05-1 liter wastewater/day per kilogramof wet wood fiber mass.
 64. The process of claim 62, wherein thewastewater is passed through wood fiber together with cellulosic fiber,plastic, powdered or ceramic media.
 65. The process of claim 49, whereinthe chemical oxygen demand of the wastewater is reduced.
 66. Awastewater treatment composition, comprising a strain of Mucorracemosus, Paecilomyces lilacinus, Aspergillus ustus or Trichodermainhamatum.